Antigenicity of infectious pancreatic necrosis virus VP2 sub-viral particles expressed in yeast

ABSTRACT

Infectious pancreatic necrosis virus (IPNV), the etiologic agent of infectious pancreatic necrosis in salmonid fish, causes significant losses to the aquaculture industry. The gene for the viral capsid protein (VP2) was cloned into a yeast expression vector and expressed in  Saccharomyces cerevisae . Expression of the capsid gene in yeast resulted in formation of approximately 20 nanometer sub-viral particles composed solely of VP2 protein. Anti-IPNV antibodies were detected in rainbow trout vaccinated either by injection of purified VP2-subviral particles (rVP2-SVP) or by feeding recombinant yeast expressing rVP2-SVP. Challenge of rVP2-SVP immunized trout with a heterologous IPNV strain and subsequent viral load determination showed that both injection and orally vaccinated fish had lower IPNV loads than naive or sham-vaccinated fish. This study demonstrates the ability of rVP2-SVPs to induce a specific immune response and the ability of immunized fish to reduce the viral load after an experimentally induced IPNV infection. The invention is not limited to IPNV, and is applicable to other similar viruses for which SVPs can be made and administered to fish.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under the provisions of 35 U.S.C. §371 andclaims the priority of International Patent Application No.PCT/US07/087,942 filed on Dec. 18, 2007, which in turn claims priorityof U.S. Provisional Application No. 60/875,901 filed on Dec. 20, 2006,the contents of which are incorporated by reference herein for allpurposes.

BACKGROUND OF THE DISCLOSURE

1. Field of Invention

The disclosure relates generally to the fields of immunology and fishproduction.

2. Related Art

Infectious pancreatic necrosis virus is the causative agent ofinfectious pancreatic necrosis disease (IPN) that infects salmonids andremains a serious problem in the aquaculture industry (1). IPN isespecially contagious and destructive to juvenile trout and salmon.Highly virulent strains may cause greater than 70% mortality in hatcherystocks over a period of two months (21). This disease is especiallydestructive in salmonid eggs and fingerlings (25). Survivors ofinfection can remain lifelong asymptomatic carriers and serve asreservoirs of infection, shedding virus in their feces and reproductiveproducts. Losses due to IPNV on salmon smoltification have beenestimated at 5% (16). Economic losses due to IPNV in aquaculture wereestimated to be over $60 million in 1996 (4), (17). This has beenreduced as vaccines for salmonids became available based on killed virusor recombinantly produced viral peptides (13, 17). However, thesevaccines are not completely effective and can only be used in fairlylarge fish due to the reliance on injection for vaccination.

IPNV is a double-stranded RNA virus of the Birnaviridae family (5) andis the type species of the Aquabirnavirus genus (6). Birnaviruses have anon-enveloped, single-shelled particle structure comprised of a singleprotein capsid layer with T=13 icosahedral symmetry (2). All birnavirusgenomes have two dsRNA segments. The IPNV genome's two dsRNA segmentsare designated segments A and B. Segment B (2777 nucleotides) encodes aminor internal polypeptide VP1 (94 kDa), which is the virion-associatedRNA-dependent RNA polymerase (RdRp) (7), (11). Segment A (3097nucleotides) encodes a 106-kDa precursor polyprotein composed ofpVP2-VP4-VP3, in that order, and a 15-kDa non-structural VP5 protein,found only in infected cells (14). VP2 and VP3 are the major capsidproteins, but VP2 is the major host-protective antigen of IPNV (9),(12).

There are commercial multivalent vaccines based on inactivated wholevirus available as well as those produced with another approach,expressing VP2-derived conserved antigenic epitopes in bacteria forproduction of a subunit vaccine. In the laboratory, these currentvaccines provide impressive protection against bath challenge with IPNV,but the behavior in the field is not predicted by the laboratorystudies. This could be due to the lack of a well-defined challengesystem with mortality as its endpoint. Results based on viral clearanceexist but may not be as rigorous as a standardized challenge model (1).Another possible explanation could be that the salmon smolts or largertrout being vaccinated are already infected with the virus, as each yearbetween 30-40% of the salmon hatcheries experience an outbreak of IPN(3) and IPN is endemic in many trout rearing areas. The need for betterfield efficacy could be achieved with improved vaccines that could beeconomically delivered to young salmonids such that subsequentvaccinations would boost existing immunity instead of trying to combatan existing acute or chronic infection.

BRIEF SUMMARY OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1, consisting of FIGS. 1A and 1B, shows pESC-ura expression vectormaps containing IPNV genes VP2 and VP3. FIG. 1A illustrates VP2 underthe GAL 10 promoter. FIG. 1B illustrates VP2 and VP3 under the GAL 10and GAL 1 promoters, respectively.

FIG. 2, consisting of FIGS. 2 A and 2B, includes images of SDS-PAGE andimmunoblot analysis of crude yeast lysates from recombinant yeast clonescontaining the IPNV VP2 gene or VP2 and VP3 genes. FIG. 2A is an imageof a Coomassie blue stained gel of IPNV infected CHSE cell culturesupernatant (+ control, lane 1), and Y-PER extracted total yeast proteinfrom the two clones expressing VP2 and VP2+VP3 (lanes 2 and 3). FIG. 2Bis an image of an immunoblot of the same samples probed with IPNVpolyclonal antibody. The molecular weights of VP2 and VP3 are 54 kDa,and 31 kDa shown by the two arrows. The VP3 band in the positive control(lane 1) was detected at a very low level and is therefore not visiblein the scanned photograph.

FIG. 3 is a transmission electron micrograph of cesium chloride gradientpurified rVP2-SVPs negatively stained with sodium phosphotungstate. Themarker bar indicates a distance of 40 micrometers.

FIG. 4, consisting of FIGS. 4A and 4B, is a pair of bar graphsindicating mean ELISA values (expressed as the absorbance of the HRPsubstrate TMB at A₄₅₀) of serum from responding fish followingimmunization with IPNV rVP2-SVPs. FIG. 4A is a graph for fish injectedwith purified rVP2-SVPs by intraperitoneal injection. The treatments forthe intraperitoneally vaccinated group include fish vaccinated withrVP2-SVPs (filled bar, n=12), adjuvant only control fish (open bar,n=8), and naive (non-immunized) fish (spotted bar, n=9). FIG. 4A is agraph for fish vaccinated orally with yeast expressing rVP2-SVPs. Thetreatments for the oral vaccinated group include fish fed dietscontaining recombinant yeast expressing rVP2-SVPs (checkered bar, n=12),non recombinant yeast (striped bar, n=1 1), and naive (non-immunized)fish (spotted bar, n=9). The error bars represent 1 SEM.

FIG. 5, consisting of FIGS. 5A and 5B, is a pair of bar graphs thatillustrate the relative load of IPNV in spleen tissue of vaccinated andnon-immunized rainbow trout as determined by SYBR Green real-timeRT-PCR. FIG. 5A shows IPNV load in rVP2-SVP injected (filled bar, n=12),and adjuvant injected (open bar, n=8) rainbow trout. FIG. 5B showsrepresents the IPNV load in rainbow trout that were orally vaccinated(diet containing yeast expressing rVP2-SVPs) (checkered bar, n=12), orcontrol fish (diet containing yeast only) (striped bar, n=11). The IPNVload was normalized with respect to rainbow trout EF-1-α expression. TheΔC_(t) values are inversely correlated to IPNV copy number. Therefore,lower the ΔC_(t) value higher the IPNV load. The error bars represent 1SEM.

DETAILED DESCRIPTION

The disclosure relates to production and care of fish, includingimmunization of fish against viral pathogens.

Here, we report cloning of the IPNV-VP2 gene into a yeast expressionvector, pESC-ura. Expression of the VP2 protein resulted in formation ofapproximately 20 nanometer sub-viral particles (SVPs) in yeast, asdetected by electron microscopy. Purified recombinant VP2 SVPs(rVP2-SVPs) were used to vaccinate fish by both injection and oralroutes and their antigenicity in rainbow trout evaluated by immunoassay.An IPNV challenge trial was also carried out and the effect ofvaccination on viral load evaluated.

An ideal vaccine for IPNV must induce long lasting protection at anearly age, prevent carrier formation, and be effective against a largenumber IPNV serotypes. Injection cannot be used for small fish,therefore either oral delivery or immersion are more preferred routesfor early vaccination. These attributes of an ideal IPNV vaccine must bemet either by a recombinant subunit vaccine or by an inactivated viralvaccine, as a live attenuated vaccine could potentially lead to carrierformation. The yeast expression system has potential value for oralvaccine development, since yeast is already a component of feeds and isgenerally regarded as safe. This contrasts with bacterial expression inEscherichia coli, which generates pyrogens that would need to be removedbefore use of any crude preparation as an oral vaccine (22). The use ofyeast is also attractive because production is economical and, throughwell-developed genetic systems, can be engineered to provide an abundantsupply of the protein or proteins of interest. In fact, Pitcovski et al.(19) reported the development and large-scale use of yeast-derivedrecombinant VP2 vaccine for the prevention of infectious bursal disease(caused by another birnavirus) of chickens.

Materials and Methods

Cloning of the VP2 and VP3 Genes of IPNV

The West Buxton (WB) strain of IPNV, obtained from American Type CultureCollection (ATCC VR-877), was used for this study. This virulent strainof IPNV is prevalent in Maine and Canada, where the major North Americansalmon aquaculture industry exists. The WB strain of IPNV was purifiedas previously described (26). The virus was propagated in Chinook salmonembryo (CHSE-214) cell cultures (ATCC CRL-1681), maintained at 15degrees Celsius in Eagle's minimal essential medium (EMEM) andsupplemented with 10% fetal bovine serum (FBS), 100 units per milliliterpenicillin, 100 micrograms per milliliter streptomycin and 1 microgramper milliliter fungizone. Total viral RNA was isolated from purifiedvirus by digesting with proteinase K (200 micrograms per milliliterfinal concentration) followed by phenol: chloroform extraction (23). TheIPNV-VP2 and VP3 genes were amplified by reversetranscription-polymerase chain reaction (RT-PCR) and cloned into thepCR2.1 vector (Invitrogen, Carlsbad, Calif.) following previouslypublished protocols (24). The primer pair used for VP2 cloning wasWBABglF (5′-GAGATCTATGAACACAACAAAGGCAACCGC-3′; SEQ ID NO: 1), containinga 5′ Bg/II site, and WBAVP2R (5′-AAGCTTAAGCCCATGTGTCCATGAC-3′; SEQ IDNO: 2), containing a 5′ HindIII site. The primer pair used to clone theVP3 gene was WBAVP3F (5′-GGATCCATGTCAGGGATGGACGAAGAACTG-3; SEQ ID NO: 3)and FA3′NCHindR (5′-ATAAGCTTGGGGGCCCCCTGGGGGGCC-3′; SEQ ID NO: 4) withBamBI or HindIII sites at the 5′ ends respectively. The integrity of theclones was verified by sequencing the plasmid DNA in both directionsusing an automated DNA sequencer (Applied Biosystems).

To make a yeast expression vector containing the VP2 gene, theVP2-containing plasmid was double digested with Bg/II and HindIII. TheVP2 fragment was gel purified, blunt-ended with Klenow enzyme, andinserted between the unique EcoRI and Bg/II sites of pESC-ura, which hadbeen blunt-ended with Klenow, behind the GAL10 promoter (FIG. 1A). Tomake the VP3 yeast vector, the VP3-containing plasmid was doubledigested with BamHI and HindIII enzymes. The VP3 fragment was gelpurified and cloned between the unique BamHI and HindIII sites ofpESC-ura behind the GAL1 promoter (FIG. 1B). Finally, to make the yeastvector that expressed both the VP2 and VP3 capsid protein genes, the VP2gene was inserted into the unique EcoRI and Bg/II sites of pESC-urabehind the GAL10 promoter in the VP3-containing constructs (FIG. 1C).

Expression of VP2 in Yeast

Yeast (Saccharomyces cerivisiae strain YH501; Stratagene, La Jolla,Calif.) were transformed using the EZ Yeast Transformation Kit (Zymed,Sari Francisco, Calif.): Mutant colonies were selected for growth onautotrophic SG-ura medium containing galactose, yeast extract withoutamino acids, and amino acid dropout mixture (all amino acids plusadenine, no uracil). Mutants were grown at 30° C. for 4 days, collectedby centrifugation, then crude protein extracts prepared using Y-PERyeast breaking buffer (Pierce Biotechnology, Rockford, Ill.). Lysateswere electrophoresed on 12% SDS-polyacrylamide gels (BioRad, Richmond,Calif.) and transferred to nitrocellulose by electroblotting. The blotswere probed with sheep-anti-IPNV polyclonal antibody (MicrotekInternational, Inc, Saanichton, B.C., Canada) and detected withrabbit-anti-sheep polyclonal antibody conjugated to HRP (BethylLaboratories, Montgomery, Tex.). Detection was obtained using thecolorimetric substrate tetramethyl benzidine (TMB) in a one stepsolution as described by the manufacturer (Pierce, Rockford, Ill.).

Isolation of rVP2-SVPs and Transmission Electron Microscopy

SVPs were isolated from yeast cultures expressing recombinant VP2according to a modified protoplasting protocol (18) to remove the yeastcell wall. The cells were lysed by three freeze thaw cycles thensonicated for five 60-second cycles with 20-second intervals. Lipidswere removed by performing two successive Freon extractions. SVPs werethen purified by passing them through a 26% sucrose cushion at 82,705×g(average) for 4 hours at 4° C. in a swinging bucket rotor (BeckmanSW28), followed by CsCl-gradient centrifugation overnight at 115,584×g(average) at 4° C. in a swinging bucket rotor (Beckman SW41). Thebuoyant density of IPNV is 1.33 grams per cubic centimeter. Bands werewithdrawn with a syringe and dialyzed overnight at 4° C. in TN buffer(50 millimolar Tris and 100 millimolar NaCl, pH 8.0) to remove CsCl.SVP's were prepared for negative staining transmission electronmicroscopy according to the previously published protocols (8). Thismethod of producing SVPs is exemplary; other methods can be used toproduce SVPs.

Immunization and Sampling of Rainbow Trout

Rainbow trout (Oncorhynchus mykiss; approximately 25 grams) originatingfrom the Clear Springs Food, Inc. (Buhl, Id.) and known to be free ofIPNV were used for the immunization experiment. The vaccination andanimal work was done at Clear Springs Foods, Inc. while the analyticalwork was performed at Advanced BioNutrition, Inc. The fish wereanesthetized and injected intraperitoneally (EP) with 100 microliters ofvaccine (50 microliters of purified rVP2-SVPs containing 100 microgramsantigen and 50 microliters of Freund's Complete Adjuvant). There werethree groups of fish: naive fish (n=9), fish injected with adjuvantsonly (sham-injected treatment; Freund's Complete Adjuvant, Sigma, St.Louis, Mo.; n=8), and a treatment group that was injected with IPNVrVP2-SVPs plus Freud's adjuvant (n=12). Vaccinations were done at days 1and 32.

For oral vaccination, recombinant yeast expressing rVP2-SVPs (withoutprior purification) was mixed with feed. Yeast were ground in liquidnitrogen then incorporated into a fish feed (Clear Spring Foods, Inc.,proprietary blend) that was first powdered using a coffee mill thensupplemented with 10% wheat gluten as binder. Feed blends were mixed byhand with moisture added as required until a pliable dough was produced.This was then fed through a press to produce ribbons of feed that werechopped to approximately 0.5 cm in length. These were allowed to air dryat room temperature for several hours then spray coated with canola oiland frozen until use. The treatments for the oral vaccination includefish that were fed diet containing yeast expressing rVP2-SVPs (n=13) ordiet containing non-recombinant yeast (control, n=10). At day 60, bloodwas withdrawn from caudal vessels of control and vaccinated fish andallowed to clot overnight at 4 degrees Celsius. Blood samples werecentrifuged in a tabletop centrifuge at 12,568×g (average) for 5minutes, then serum was collected and stored at −75 degrees Celsiusuntil analyzed.

Enzyme-Linked Immunosorbent Assay (ELISA)

Immuno Breakapart microplates (Nunc, Rochester, N.Y.) were coated withpurified IPNV rVP2-SVPs at 150 micrograms per milliliter in a 50millimolar carbonate coating buffer (pH 9.6) at 4° C. for 16 hours.Plates were washed 3 times in TBST (IX Tris Buffered Saline (TBS)+0.05%Tween 20) for 5 minutes each wash. The plates were blocked with Ix TBScontaining 3% BSA at room temperature. Test sera were diluted 1:32 and1:64 then 150 microliters was added per well and the plates wereincubated for 1 hour at room temperature. Following incubation with testsera, the microplates were washed again 3 times with TBST for 5 minutesper wash. The secondary antibody (rabbit anti-rainbow trout IgG; JacksonImmunoResearch Laboratories Inc, West Grove. Pa.) was diluted 1:1000 andadded to all wells (150 microliters/well). The plates were incubated for1 hour at room temperature and then washed 3 times in TBST, 5 minuteseach wash. Horseradish peroxidase-conjugated goat anti-rabbit IgG(Biosource, Camarillo, Calif.) was added at a 1:1.000 dilution anddetected by addition of the colorimetric substrate tetramethyl benzidine(TMB, Pierce, Rockford, Ill.). The absorbance was read at 450 nanometersusing a SPECTRAFLUOR® Plus fluorescent plate reader (Tecan, Salzburg,Austria). Negative controls consisted of wells that were coated asabove, but a 3% BSA solution was added instead of the fish serum at thecapture step.

IPNV Challenge and Sample Collection

Three days after collecting the blood samples (i.e., at 63 dayspost-vaccination), IPNV challenge was performed by injecting each fishwith approximately 250 microliters of 10⁷ TCID₅₀/mL of IPNV (Buhlstrain, LaPatra unpublished). Naive fish injected with buffer served asnegative control for the IPNV challenge. Ten days post-injection,animals were sacrificed, spleen samples collected in TR1 reagent, thenstored at −75 degrees Celsius until RNA isolation was performed.

Isolation of Total RNA and cDNA Synthesis

Total RNA was isolated from spleen tissue of control and IPNV-injectedrainbow trout using TRI reagent following the manufacturer's protocols(Molecular Research Center, Cincinnati, Ohio). The RNA samples weretreated with DNase I (Ambion, Enc, Austin, Tex.) then the RNA qualityassessed by running the samples on a 1% formaldehyde agarose gel (23).The cDNA synthesis was carried out in a 40 microliter reaction volumecontaining 1 microgram total RNA, Ix RT-PCR buffer. 1 millimolar dNTPs,0.75 micromolar oligo dT, 4 units of RNase inhibitor, and 5 units ofMultiScribe reverse transcriptase (Applied Biosystems, Foster City,Calif.) at 42 degrees Celsius for 1 hour. The cDNA was diluted 1:10using DNase and RNase free molecular biology grade water and 2microliters of the diluted cDNA was taken for each reaction.

Determining IPNV Load by SYBR Green Real-Time RT-PCR

The primers for the SYBR Green real-time RT-PCR were designed based onthe nucleotide sequence of segment A of the IPNV genome that encodes theprotease protein (VP4) (GenBank Accession no. NC_(—)001915, forwardprimer 1916F: 5′ AGGAGATGAC ATGTGCTACACCG 3′; SEQ ID NO: 5, and reverseprimer 1999R: 5′CCAGCGAATA TTTTCTCCACCA 3′; SEQ ID NO: 6). The rainbowtrout elongation factor 1-α (EF-1-α) gene was used as an internalcontrol for normalizing the viral load from sample to sample. Theprimers for rainbow trout elongation factor 1-α (EF-1-α) were based onthe published sequence of these genes (GenBank Accession no AF498320,forward primer 136F: 5′ TGATCTACAAGTGCGGAGGCA 3′; SEQ ID NO: 7, andreverse primer 236R: 5′ CAGCACCCAGGCATACTTGAA 3′; SEQ ID NO: 8). Theprimers were designed using the Primer Express Software version 1.0(Perkin Elmer-Applied Biosystem). The real-time RT-PCR amplificationswere performed in a BioRad iCycler iQ (BioRad Laboratories, Inc.,Richmond, Calif.).

The SYBR Green real-time RT-PCR mixture contained 12.5 microliters of 2×SYBR. Green Supermix (iQ SYBR Green Supermix), 300 nanomolar each offorward and reverse primers and 2 microliters of the 1:10 diluted cDNAin a 25 microliters reaction volume. The amplifications were carried outin a 96-well microplate with 3 replicates per sample. The thermalprofile for SYBR Green real-time RT-PCR was 95 degrees Celsius for 10minutes, followed by 40 cycles of 95 degrees Celsius for 10 seconds and60 degrees Celsius for 1 minute.

After a SYBR Green PCR run, data acquisition and subsequent dataanalyses were performed using the iCycler iQ Real-Time PCR DetectionSystem (BioRad iQ Software Version 1.3). The relative IPNV load in asample was determined by subtracting the mean C_(t) values for EF-1αfrom the mean C_(t) values of the IPNV amplicon. The differences in theC_(t) value of the viral genes and the corresponding internal controlswere expressed as ΔC_(t). The ΔC values were plotted using GraphPadVersion 4 (Graphpad Software, Inc., San Diego, Calif.). The differencein the ΔC_(t) for one vaccine group compared to the ΔC_(t) of thecorresponding control was expressed as a ΔΔC_(t) and 2 represents thedifference in viral load between the two treatments.

Results and Discussion

The IPNV segment A has previously been cloned and expressed in hamsterfibroblast cells, BHK-21, under the Semliki forest virus promoter and ininsect cells under the polyhedrin promoter (polh) and were shown toproduce virus-like particles (VLPs) that contain both VP2 and VP3 andare of similar size to the native virus but lack associated nucleic acid(15), (24). However, when we cloned the IPNV segment A in yeast, thepolyprotein was expressed but no particles were observed under TEM (datanot shown). This might be due to the lack of post-translationalprocessing of the polypeptide in yeast. Therefore, we coexpressed VP2and VP3 genes under different promoters into the pESC-ura vector so thatthe post-translational processing of the polyprotein would not berequired. For clarity in the following discussion, the authors use theterm virus-like particle (VLP) to describe viral-derived particles ofsimilar size to the native virus that lack nucleic acid. For particlesthat are viral-derived and lack nucleic acid but do not have the samesize or shape as the native virus the authors use the term sub-viralparticle (SVP) to differentiate the two sets of viral-derived particles.

Cloning of VP2 and VP3 Genes

The predicted mature VP2 and VP3 genes were cloned separately behindGAL10 and GAL1 promoters in pESC-ura. Recombinant yeast containing VP2or both VP2 and VP3 genes were grown under galactose induction thenanalyzed by western blot analysis to determine if VP2 and VP3 wereexpressed (FIG. 2). Two bands were observed that corresponded roughly tothe molecular weights predicted for VP2 and VP3 in the co-expressionsystem, 54 kDa and 31 kDa respectively (FIG. 2, right panel). The immuneblots indicated the presence of both VP2 and VP3 in our yeast mutantdesigned to express both genes when grown under galactose induction.

Preparation of SVPs and/or VLPs Plus Subsequent Electron Microscopy

Using the methods described above, VLP or SVP preparations were preparedon the clones containing both VP2 & VP3 genes. Several areas of highdensity were observed in the CsCl gradients. The high molecular weightmaterials pelleted in the ultracentrifuge, and a band of moderatedensity was observed in the gradient. The moderate density bandcorresponded to a approximately 20 nm particle that contained only VP2reacting materials (FIG. 3). However, 60 nm full sized IPNV virus-likeparticles, as seen previously in IPNV segment A expression in insectcells (24), were not observed. Similar particles have been previouslydescribed for IPNV (10) and are thought to be due to an error in pVP2processing. Similar particles were also observed and characterized inIBDV (20). They are formed by 20 VP2 subunit trimers in a T=1 fashion.VP3 is not involved in their formation. Here, we saw the same thingwhether VP2 was expressed in yeast simultaneously with the VP3 gene oralone in yeast. These particles are referred to herein as sub-viralparticles (SVPs). Similar methods can be used to produce SVPs forsimilar viruses (e.g., other viruses having capsid proteins from whichSVPs can be formed, such as other Birnaviridae family viruses and otherviruses for which salmonids or other fish are a host). The compositionsdescribed herein can be produced by a variety of methods available toskilled worker in this field, and SVPs made by any of these methods areexpected to be useful in the methods described herein.

Immunization of Rainbow Trout

Rainbow trout that were free of IPNV were used for a vaccinationexperiment testing both intraperitoneal injection (IP) with adjuvant andby oral delivery in feed. The rVP2-SVPs were delivered either aspurified SVPs (for IP injection) or as crude yeast lysate incorporatedinto feeds (for oral delivery) to test the antigenicity of these IPNVsubunit vaccines in particle form in rainbow trout. The experimentaldesign is outlined in Table 1. To test the ability of rVP2-SVPs toinduce anti-IPNV antibody production, the most direct method is to usepurified antigen and deliver by injection. Purified rVP2-SVPs weredelivered by IP injection with Freud's adjuvant as described in Tables 1and 2. A booster of the same composition was delivered after 32 days andfish bled at 63 days. All of the injected fish had significantly highertiters of anti-IPNV antibodies than either the naive or sham-injectedcontrols (FIG. 4A). The naive fish and the sham-injected fish were notsignificantly different from each other at the 95% confidence intervalwhen compared using the student's t-test. The purified rVP2-SVP injectedfish showed 100% seroconversion (Table 2; FIG. 4A). Student's t-testswere run in Statview Version 5.01 (SAS Institute, Inc.), testing forsignificant differences between antibody titers of vaccine injected orfed animals compared to both the naive fish and sham-injected fish(negative controls). At the 1:32 serum dilution, the rVP2-SVP injectedfish had a significantly higher seroconversion rate when compared to thenaive fish (p=0.013) and the sham-injected fish (p=0.001). The 1:64serum dilution also demonstrated significant seroconversion differencesbetween rVP2-SVP injected fish and negative controls (p=0.0003, naivefish and p=0.0007, sham-injected fish).

Oral vaccination would provide a number of advantages over injectionsuch as ease of use, ability to vaccinate smaller fish, lower cost ofvaccine, and easy ability to make multivalent vaccines (through deliveryof different clones in the feeds). In order to test the ability ofrVP2-SVPs to induce an immune response, recombinant yeast expressingVLPs were incorporated into fish feed and fed to one treatment group forseven days. At day 32 another seven day feeding of the recombinant yeastcontaining feed was done as a booster (Table 1). At 63 days the fishwere bled and the anti-IPNV titers compared to that found in naive fishand fish fed a control feed supplemented with wild-type yeast in placeof the recombinant yeast (FIG. 4B). It was apparent that the orallyvaccinated fish had an immune response greater than that observed ineither naive or yeast control fed fish (p=0.0002 for naive fish andp=0.0053 for yeast control). There appeared to be a higher anti-IPNVtiter in the yeast control sera than in the naive fish, but thedifference was not significant (p=0.1645) as determined by the studentt-test. Seroconversion of the orally vaccinated fish was slightly lessthan that observed in the IP injected animals with approximately 75%conversion (Table 2). Oral vaccination with rVP2-SVPs provides anincrease, albeit reduced relative to IP injection, in anti-IPNV titer.

While these data do not demonstrate the effectiveness of thesevaccination strategies on prevention of disease, they are an indicationthat oral vaccination could potentially provide an alternative to IPinjection vaccination for the treatment of IPN. A challenge trial wouldprovide definitive evidence that this approach could prevent disease.

The results presented herein pertain to rainbow trout. However, themethods and compositions are not so limited in their applicability. Oneexpects such compositions and methods to be effective in other types offish as well, including not only salmonids. Furthermore, the efficacy ofthe compositions described herein for enhancing immunity and preventingdisease are not limited to the methods of administration that areexplicitly described herein. Other methods of administering immunogeniccompositions to fish are expected to yield similar efficacy.

IPNV Challenge/Viral Load

There is no good challenge system for IPNV with mortality as theendpoint (1). Using IPNV viral load, as determined by real-time RT PCR,could provide a convenient method to track the progress of the disease.In this study, the trout were vaccinated with either rVP2-SVPs deliveredin feed or by injection of purified rVP2-SVPs derived from the WestBuxton strain of IPNV. After 63 days post-vaccination, fish wereinjected with the Buhl strain of IPNV that had been isolated fromrainbow trout in Idaho (La Patra, unpublished data). This was adifferent IPNV strain (Buhl) than that from which the rVP2-SVPs vaccinewas derived (West Buxton strain). Therefore, the challenge was with aheterologous strain and may help evaluate the specificity of thisapproach. IP vaccinated rainbow trout had significantly less virus(p=0.0280) (22 fold) than sham-injected control fish (Table 3, FIG. 5).When oral vaccinates were compared to the yeast only controls, a 12-foldreduction in virus was found for IPNV vaccinated fish (FIG. 5B). Thisdifference was visually apparent, but not significant at the 0.05 level(p=0.1179).

These data indicate that rVP2-SVPs produced in yeast could provide anovel means for amplification of a protective immune response in rainbowtrout, and by extension to salmonid species like salmon, either byinjection or by delivery in feeds. Expression of a rVP2-SVP particle inyeast provides an interesting opportunity for its use as a vaccine fortrout and salmon. The ability of these particles to induce theproduction of IPNV-specific antibodies was demonstrated by both oral andinjection routes. The potential for use of the oral route as a vaccineneeds further investigation to optimize the immune response anddetermine if the observed decrease in viral load directly correlateswith prevention of IPN. This study sets the foundation for furtherstudies to test in juvenile salmonids the utility of this approach toprevent early onset of IPN.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

While this subject matter has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations can bedevised by others skilled in the art without departing from the truespirit and scope of the subject matter described herein. The appendedclaims include all such embodiments and equivalent variations.

REFERENCES

Literature referred to herein is as follows:

-   (1) Biering, E., S. Villoing, I. Sommerset, K. E. Christie. 2005.    Update on viral vaccines for fish In: P. J. Midtlyng (ed.), Progress    in Fish Vaccinology. Dev. Biol. Basel 121:97-113.-   (2) Botteher, B., N. A. Kiselev, V. Y. Stel'Mashchuk, N. A.    Perevozchikova, A. V. Borisov, and R. A. Crowther. 1997.    Three-dimensional structure of infectious bursal disease virus    determined by electron cryomicroscoopy. J Virol 71(1): 325-330.-   (3) Brun, E. 2003. Epidemiology. In: O. Evensen, E. Rimstad, R.    Stagg, E. Brun, P. Midtlyng, B. Skjelstad. L. H. Johansen, and I.    Jensen (eds.), IPN in salmonids: a review. FHL & VESO, Trondheim,    Norway, pp. 51-67.-   (4) Christie, K. E. 1997. Immunization with viral antigens:    infectious pancreatic necrosis. Dev. Biol. Stand. 90: 191-199.-   (5) Delmas, B., Kibenge, F. S. B., Leong, J. A., Mundt, E.,    Vakharia, V. N., Wu, J. L., 2005. Birnaviridae, p. 561-569. In C. M.    Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, and A. L. Ball    (ed.), Virus Taxonomy. Academic Press, London, U.K.-   (6) Dobos, P., 1995. The molecular biology of infectious pancreatic    necrosis virus (IPNV). Ann. Rev. Fish Dis. 5, 24-54.-   (7) Duncan, R., Mason, C. L, Nagy, E., Leong, J. A., Dobos,    P., 1991. Sequence analysis of infectious pancreatic necrosis virus    genome segment B and its encoded VP1 protein: a putative    RNA-dependent RNA polymerase lacking the Gly-Asp-Asp motif. Virology    181(2), 541-552.-   (8) Dykstra M J. (1.992) Specimen preparation for transmission    electron microscopy. In Dykstra M J. ed. Biological Electron    Microscopy. New York, London, Plenum Press, 5-78.-   (9) Frost P., L. S. Havarstein, B. Lygren, S. Stahl, C.    Endresen, K. E. Christie. 1995. Napping of neutralization epitopes    on infectious pancreatic necrosis viruses. J. Gen. Virol. 76 (Pt 5):    1165-1172.-   (10) Galloux, M., C. Chevalier, C. Henry, J.-C. Huet, B. Da    Costa, B. Delmas 2004. Peptides resulting from the pVP2 C-terminal    processing are present in infectious pancreatic necrosis virus    particles. J. Gen. Virol. 85(Pt 8): 2231-2236.-   (11) Gorbalenya. A. E., F. M. Pringle, J. L. Zeddam, B. T.    Luke, C. E. Cameron, J. Kalmakoff, T. N. Hanzlik, K. H. Gordon,    and V. K. Ward. 2002. The palm subdomain-based active site is    internally permuted in viral RNA-dependent RNA polymerases of an    ancient lineage. J. Mol. Biol. 324 (1): 47-62.-   (12) Heppell J., E. Tarrab, J. Lecomte, Berthiaume, and M.    Arella. 1995. Strain variability and localization of important    epitopes on the major structural protein (VP2) of infectious    pancreatic necrosis virus. Virology 214 (1): 40-49.-   (13) Labus, M. B., S. Breeman, A. E. Ellis, D. A. Smail, M. Kervick    and W. T. Melvin. 2001. Antigenic comparison of a truncated form of    VP2 of infectious pancreatic necrosis (IPN) virus expressed in four    different cell types. Fish & Shellfish Immunology 11(3): 203-216.-   (14) Magyar, G. and P. Dobos. 1994. Evidence to the detection of the    infectious pancreatic necrosis virus polyprotein and the 17 kDa    polypeptide in infected cells and the NS protease in purified virus.    Virology 204(2): 580-589.-   (15) McKenna, B. M., R. M. Fitzpatrick, K. V. Phenix, D. Todd, L. M.    Vaughan and G. J. Atkins. 2001. Formation of infectious pancreatic    necrosis virus-like particles following expression of segment A by    recombinant semliki forest virus. Marine Biotechnology 3(2):    103-110.-   (16) Melby, H. P., Caswell-Reno, and K. Falk. 1994. Antigenic    analysis of Norwegian aquatic birnavirus isolates uwin monoclonal    antibodies J. Fish Dis. 17: 85-91.-   (17) Midtlyng, P. 2003. Vaccination. In: Evensen O, Rimstad E, Stagg    R, Brun E, Midtlyng P., Skjelstad B. Johansen L H, Jensen I (eds.),    IPN in salmonids: a review. FHL & VESO, Trondheim, Norway, pp.    85-95.-   (18) Pannunzio, V. G., Burgos, H. I., Alonso, M., Ramos, E. H.,    Mattoon, J. R., Stella, C A. 2004. Yeast Plasmids with the Least    Trouble. Promega Notes #87: 27-28.-   (19) Pitcovski, J., B. Gutter, et al. (2003). “Development and    large-scale use of recombinant VP2 vaccine for the prevention of    infectious bursal disease of chickens.” Vaccine 21(32): 4736-43.-   (20) Pous, J., C. Chevalier, M. Ouldali, J. Navaza, B. Delmas and J.    Lepault. 2005. Structure of birnavirus-like particles determined by    combined electron cryomicroscopy and X-ray crystallography. J. Gen.    Virol. 86(Pt 8): 2339-2346.-   (21) Roberts, R. J. and M. D. Pearson 2005. Infectious pancreatic    necrosis in Atlantic salmon, Salmo salar L. J. Fish Diseases 28(7):    383-390.-   (22) Romanos, M. A., C. A. Scoer, and J. J. Clare. 1992. Foreign    gene expression in yeast: a review. Yeast 8(6): 423-488.-   (23) Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular    cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory,    Cold Spring Harbor, N.Y.-   (24) Shivappa, R. B., P. E. McAllister, G. H. Edwards, N. Santi, O.    Evensen, and V. N. Vakharia. 2005. Development of a subunit vaccine    for infectious pancreatic necrosis virus using a baculovirus    insect/larvae system. In: P. J. Midtlyng (ed.) Progress in Fish    Vaccinology. Dev. Biol. Basel 121: 165-174.-   (25) WolfK., S. Snieszko, C. Dunbar, E. Pyle. 1960. Virus nature of    infectious pancreatic necrosis in trout. Proc. Soc. Exp. Biol. Med.    104: 105-108.-   (26) Yao, K. and V. N. Vakharia. 1998. Generation of infectious    pancreatic necrosis virus from cloned cDNA. J. Virol. 72(11):    8913-8920.

TABLE 1 Experimental design for vaccination trial on rainbow trout withIPNV rVP2-SVPs delivered by either intraperitoneal injection (IP) ororally in feeds. Vaccine delivery No. of (Injection/feeding)* Animals/Treatment Initial (Day) Booster (Day) Treatment Naïve fish None None 9(Day 1) (Day 32) Injection Control Injection Injection 8 (Day 1) (Day32) Injected rVP2-SVPs Injection Injection 12 (Day 1) (Day 32) ControlYeast Oral Oral 10 (Days 1-7) (Days 32-38) Oral rVP2-SVPs Yeast OralOral 13 (Day 1-7) (Days 32-38)

TABLE 2 Effect of intraperitoneal or oral vaccination with IPNVrVP2-SVPs on the titer of anti-IPNV antibodies in rainbow trout. VaccineSerum Mean A₄₅₀ Seropositives/ Treatments* delivery Dilution value ± SEMTotal Number** Naive fish None 1:32 0.263 ± 0.022 0 1:64 0.235 ± 0.023 0Adjuvant Injection 1:32 0.363 ± 0.049 0 Control 1:64 0.232 ± 0.037 0rVP2-SVPs Injection 1:32 0.982 ± 0.128 12 of 12 1:64 0.701 ± 0.090 12 of12 Control yeast Oral 1:32 0.346 ± 0.035 0 1:64 0.295 ± 0.026 0rVP2-SVPs Oral 1:32 0.530 ± 0.045 10 of 13 Yeast 1:64 0.414 ± 0.034  9of 13 *Naive fish were not injected and were fed normal fish feed,adjuvant control fish were IP injected with buffer and adjuvant,rVP2-SVPs fish were injected with 100 μg of antigen plus adjuvant.control yeast fish were fed fish feed supplemented with wild-type yeast,and rVP2-SVPs yeast fish were fed fish feeds containing the recombinantyeast. **Fish considered seropositive if A₄₅₀ was above the meanadjuvant control plus one standard error.

TABLE 3 Relative quantification of IPNV load by real-time RT-PCR inrVP2-SVP vaccinated rainbow trout. IPNV Fold Vaccine Delivery Averagereduction Treatments Injection/Feeding ΔCt* ΔΔ Ct** (2^(ΔΔ Ct)) AdjuvantInjection Injection 9.27 control (Day 1) (Day 32) rVP2-SVPs InjectionInjection 13.75 4.49 22.40 (Day 1) (Day 32) Control Oral Oral 5.22 Yeast(Days 1-7) (Days 32-38) rVP2-SVPs Oral Oral 8.83 3.61 12.25 Yeast (Days1-7) (Days 32-38) *ΔCt was first calculated for each fish using the Ctvalues of IPNV for a fish minus the Ct values of EF-I alpha gene for thesame fish. Then the average ΔCt was calculated taking the Ct value ofall the fish in each treatment. **ΔΔ Ct = Average ΔCt value of atreatment minus the average ΔCt value of the corresponding controltreatment

1. A composition comprising aquatic species feed and a crude yeast lysate, wherein the crude yeast lysate comprises a recombinant yeast expressing Infectious Necrosis Virus segment A protein consisting of capsid protein 2 (VP2) as recombinant VP2 sub-viral particles (rVP2-SVPs), wherein the rVP2-SVPs are not purified from the yeast and wherein the rVP2-SVPs are about 20 nanometers in diameter size.
 2. The composition according to claim 1, wherein the aquatic species feed is fish food for oral administration to fish for controlling Infectious Pancreatic Necrosis Virus (IPNV) therein.
 3. The composition according to claim 1, wherein the yeast does not generate pyrogens that must be removed before administration to an aquatic species.
 4. An oral vaccine for controlling Infectious Pancreatic Necrosis Virus (IPNV), wherein the oral vaccine comprises the recombinant yeast according to claim
 1. 5. The oral vaccine according to claim 4, wherein the recombinant yeast was mixed with aquatic feed for feeding to an aquatic species.
 6. A method to enhance immunity against IPNV comprising: administering an effective amount of the composition according to claim 1 to aquatic species for a period of at least seven days.
 7. A method of generating a composition comprising aquatic species feed and a crude yeast lysate comprising the steps of: (a) providing a recombinant yeast expression vector comprising a polynucleotide encoding IPNV Segment A proteins consisting of capsid protein 2, VP2; (b) transfecting yeast with the recombinant yeast expression vector; and (c) maintaining suitable conditions for expression of rVP2-sub-viral particles (SVPs) of Infectious Pancreatic Necrosis Virus (IPNV); (d) harvesting the yeast that expressed rVP2-SVPs; and (e) combining the yeast that expressed rVP2-SVPs with the aquatic species feed, wherein the rVP2-SVPs are not purified or isolated from the yeast and wherein the rVP2-SVPs are about 20 nanometers in diameter size.
 8. The method according to claim 7, wherein the aquatic species feed is fish food for oral administration to fish for controlling IPNV therein. 